In this revised application, we will primarily focus on studying the role of NO and cyclic GMP signaling in proliferation and differentiation of human and mouse embryonic stem cells into cardiomyocytes. Our preliminary studies strongly suggest a role for nitric oxide and cyclic GMP in mouse and human embryonic stem cell proliferation and differentiation. The mRNA (RT-PCR and Northerns) and protein (Western and activity) in mouse and human stem cells (WA09) will be examined during stem cell proliferation and differentiation for nitric oxide synthases (NOS 1,2,3) and soluble guanylyl cyclase, the receptor for NO. The cellular localization will be confirmed with immunohistochemistry for these proteins and cellular markers and cell sorting. The effects of activators and inhibitors of NOS, soluble guanylyl cyclase, protein kinase G, and cyclic nucleotide phosphodieserases will also be examined to confirm the role of NO and cGMP in stem cell proliferation and differentiation into myocardial cells. With an understanding of the presence or absence of the proteins participating in nitric oxide and cyclic GMP synthesis and function in stem cells and differentiated cells with their in vitro culture, we expect to be able to design experimental protocols with various pharmacologic agents such as NO donor drugs, cyclic nucleotide analogues, phosphodieterase inhibitors, and guanylyl cyclase activators that should modify stem cell proliferation and differentiation into various cellular lineages. The function of these stem cells and differentiated cells will also be examined in several cardiovascular models. This should be extremely valuable information for future clinical studies by others and us for the purpose of tissue replacement and perhaps drug and gene delivery systems. Several colleagues with expertise in stem cell biology and/or in vivo animal models will serve as advisors, consultants, and collaborators. Our preliminary experiments and the work of others indicate that our studies proposed are feasible and are likely to be successful to determine the roles of nitric oxide and cyclic GMP in stem cell proliferation, differentiation, and function.